1. Analyzes of hereditary abnormal molecules of blood coagulation and fibrinolysis. 1) Hereditary dysfibrinogenemias : During 1992-1993, we have completed a study on two dysfibrinogens, fibrinogen (Fbg) Bremen (Aalpha Gly-17 to Val) and Fbg Mitaka II(Aalpha Glu-11 to Gly). Fbg Bremen had been found in a 14-year-old German boy with surgical bleeding and delayd wound healing, and the blood sample was sent to our laboratory for structure analysis. The Aalpha Gly-17 to Val substitution provided supporting evidence that the amino terminal three-residue peptide of fibrin alpha-chain, Gly-Pro-Arg, constitutes a polymerization site, "A" exposed onto the central domain of fibrinogen. By utilizing synthetic peptides with a normal or a Bremen type sequence, or their related sequences, we have shown that the free amino group of the amino-terminal residue of fibrin alpha-chain is most critical for the function of the "A" site, since the replacement of Gly to Ala or Val manifested only a minute func
… Moretional disturbance, whereas that to other amino acids failed to manifest polymerization-promoting activity (Publication No. 15). Fbg Mitaka II was characterized by defective binding with thrombin. Thus Aalpha Glu-11 appears to play a crucial role in the binding with thrombin. indeed, the side-chain carboxy group of this amino acid has recently been shown by X-ray crystallography to form a salt bridge with the side-chain guanidino group of Arg-173 of thrombin, and also to stabilize the type II beta-turn which is mandatory for fibrinogen to be fitted into the enzyme pocket of thrombin. Our data support this hypothesis, and provide important implications in the study of thrombus formation (publications No. 13 and 15). 2) An abnormal factor IX Tokyo I found in a mild hemophiliac : By gene analysis, we have identified a T to C mutation at nucleotide 20525 coding for Val-182 of factor IX.Thus the patient's factor IX must have an ala-182 substitute. This site is close to the cleavage site by2. Study on the cell adhesion : High-molecular-weight kininogen was found to manifest cell attachment promoting activity when converted to a two-chain molecule (HKa). We have been analyzing the molecular basis for this phenomenon by utilyzing a battery of synthetic peptides and monoclonal antibodies produced by the support of this research fund. We have also identified specific integrins on cultured glioma cells upon contact with vitronectin and fibrinonectin, which are normally absent but exposed upon contact with these adhesion molecules. Interestingly these integrins appear to migrate on the cell surface in accordance with the cell shape changes (submitted to Brain Research).2)軽症血友病Bを惹起する異常第IX因子:Tokyo I:活性型第XIまたは第VII因子による活性化に際して開裂される部位近傍にVal-182→Ala置換を遺伝子解析で同定するとともに、これに符号する活性化遅滞の分子機作を精製異常IX分子を用いて酵素化学的に解明し、報告した(業績14)。2.細胞接着に関する研究:ヒト高分子キニノゲンが2本鎖に開裂すると細胞接着を阻害することからその分子機作を種々の合成ペプチドやモノクロナル抗体を用いて解析中である。別に、ヒトglioma cellでintegrinの発現と分布を血漿中の各種接着分子との接着反応系で観察した。その結果、生理的環境下には発現しないαVβ3とα5β1の発現および細胞膜上での特異な分布とその移動を見出し、Brain Researchに投稿中である。 Less